title: High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.
abstract: G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.
authors.affiliation: Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Bing
authors.initials: B
authors.lastname: Xu
authors.forename: Raja
authors.initials: R
authors.lastname: Chakraborty
authors.forename: Markus
authors.initials: M
authors.lastname: Eilers
authors.forename: Shyamala
authors.initials: S
authors.lastname: Dakshinamurti
authors.forename: Joe D
authors.initials: JD
authors.lastname: O'Neil
authors.forename: Steven O
authors.initials: SO
authors.lastname: Smith
authors.forename: Rajinder P
authors.initials: RP
authors.lastname: Bhullar
authors.forename: Prashen
authors.initials: P
authors.lastname: Chelikani
mesh_terms: D000595:Amino Acid Sequence; D003902:Detergents / Q000494:pharmacology; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011110:Polymorphism, Genetic; D017433:Protein Structure, Secondary; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D003902:Detergents; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013752:Tetracycline